Fiber-optics based fluorescence detection. Part I: Basic concepts.

Continuous in-line detection and process monitoring are essential for industrial, analytical, and biomedical applications. Lightweight, highly flexible, and low-cost fiber optics enable the construction of compact and robust hand-held devices forin situchemical and biological species analysis in both industrial and biomedicalin vitro/in vivodetection. Despite the broad range of fiber-optic based applications, we lack a good understanding of the parameters that govern the efficiency of light collection or the sensitivity of detection. Consequently, comparing samples of different optical density and/or geometry becomes challenging and can lead to misinterpretation of results; especially when we lack the approaches necessary to correct the detected signal (spectra) for artifacts such as inner-filter effect or scattering. Hence, in this work, we discuss factors affecting the signal detected by the fiber optic in the bare and lens-coupled flat-tipped configurations that lead to signal/spectral distortions. We also present a simple generic model describing the excitation profile and emission collection efficiency that we verify with experimental data. Understanding the principles governing the signal collected by the fiber will provide rationales for correcting the measured emission spectra and recovering the true emission profile of optically dense samples.

Multi intercalators FRET enhanced detection of minute amounts of DNA.

A novel approach is presented that increases sensitivity and specificity for detecting minimal traces of DNA in liquid and on solid samples. Förster Resonance Energy Transfer (FRET) from YOYO to Ethidium Bromide (EtBr) substantially increases the signal from DNA-bound EtBr highly enhancing sensitivity and specificity for DNA detection. The long fluorescence lifetime of the EtBr acceptor, when bound to DNA, allows for multi-pulse pumping with time gated (MPPTG) detection, which highly increases the detectable signal of DNA-bound EtBr. A straightforward spectra/image subtraction eliminates sample background and allows for a huge increase in the overall detection sensitivity. Using a combination of FRET and MPPTG detection an amount as small as 10 pg of DNA in a microliter sample can be detected without any additional sample purification/manipulation or use of amplification technologies. This amount of DNA is comparable to the DNA content of a one to two human cells. Such a detection method based on simple optics opens the potential for robust, highly sensitive DNA detection/imaging in the field, quick evaluation/sorting (i.e., triaging) of collected DNA samples, and can support various diagnostic assays.

Fluorescence measurements: importance of G-factor correction, magic angle, and observation wavelengths.

Excitation and emission (observation) conditions heavily impact fluorescence measurements. Both observed spectra and intensity decays (fluorescence lifetimes), when incorrectly measured, may lead to incorrect data interpretations. In this report, we discuss the role of observation conditions in steady-state and time-resolved (lifetime) fluorescence measurements. We demonstrate the importance of the correction for uneven transmissions of vertical and horizontal polarizations of emission light through the detection system. The necessity of using so-called total fluorescence intensity or intensity measured under magic angle (MA) conditions has been demonstrated for both steady-state and time-resolved fluorescence measurements. The dependence of lifetime measurements on observation (emission) wavelengths is also discussed. Two fluorophores, rhodamine 6G (R6G) and 4,4 Dimethylamino-cyano stilbene (DCS) in two solvents - ethanol and glycerol have been used in order to cover a broad range of dye polarities and solvent viscosities.

Imaging and detection of long-lived fluorescence probes in presence of highly emissive and scattering background.

Optical biomedical imaging and diagnostics is a rapidly growing field that provides both structural and functional information with uses ranging from fundamental to practical clinical applications. Nevertheless, imaging/visualizing fluorescence objects with high spatial resolution in a highly scattering and emissive biological medium continues to be a significant challenge. A fundamental limiting factor for imaging technologies is the signal-to-background ratio (SBR). For a long time to improve the SBR, we tried to improve the brightness of fluorescence probes. Many novel fluorophores with improved brightness (almost reaching the theoretical limit), redshifted emission, highly improved photostability, and biocompatibility greatly helped advance fluorescence detection and imaging. However, autofluorescence, scattering of excitation light, and Raman scattering remain fundamental limiting problems that drastically limit detection sensitivity. Similarly, significant efforts were focused on reducing the background. High-quality sample purification eliminates the majority of autofluorescence background and in a limited confocal volume allows detection to reach the ultimate sensitivity to a single molecule. However, detection and imaging in physiological conditions does not allow for any sample (cells or tissue) purification, forcing us to face a fundamental limitation. A significant improvement in limiting background can be achieved when fluorophores with a long fluorescence lifetime are used, and time-gated detection is applied. However, all long-lived fluorophores present low brightness, limiting the potential improvement. We recently proposed to utilize multipulse excitation (burst of pulses) to enhance the relative signal of long-lived fluorophores and significantly improve the SBR. Herein, we present results obtained with multipulse excitation and compare them with standard single-pulse excitation. Subtraction of images obtained with a single pulse from those obtained with pulse burst (differential image) highly limits background and instrumental noise resulting in more specific/sensitive detection and allows to achieve greater imaging depth in highly scattering media, including skin and tissue.

Förster Resonance Energy Transfer-Enhanced Detection of Minute Amounts of DNA.

This article presents a novel approach to increase the detection sensitivity of trace amounts of DNA in a sample by employing Förster resonance energy transfer (FRET) between intercalating dyes. Two intercalators that present efficient FRET were used to enhance sensitivity and improve specificity in detecting minute amounts of DNA. Comparison of steady-state acceptor emission spectra with and without the donor allows for simple and specific detection of DNA (acceptor bound to DNA) down to 100 pg/µL. When utilizing as an acceptor a dye with a significantly longer lifetime (e.g., ethidium bromide bound to DNA), multipulse pumping and time-gated detection enable imaging/visualization of picograms of DNA present in a microliter of an unprocessed sample or DNA collected on a swab or other substrate materials.

Direct excitation of tryptophan phosphorescence. A new method for triplet states investigation.

We studied room temperature phosphorescence of tryptophan (TRP) embedded in poly (vinyl alcohol) films. With UV (285 nm) excitation, the phosphorescence spectrum of tryptophan appears at about 460 nm. We also observed the TRP phosphorescence with blue light excitation at 410 nm, well outside of the S0→S1absorption. This excitation reaches the triplet state of tryptophan directly without the involvement of the singlet excited state. The phosphorescence lifetime of tryptophan is in the sub-millisecond range. The long-wavelength direct excitation to the triplet state results in high phosphorescence anisotropy which can be useful in macromolecule dynamics study via time-resolved phosphorescence.

On the origin and correction for inner filter effects in fluorescence. Part II: secondary inner filter effect -the proper use of front-face configuration for highly absorbing and scattering samples.

Fluorescence is an established technology for studying molecular processes and molecular interactions. More recently fluorescence became a leading method for detection, sensing, medical diagnostics, biotechnology, imaging, DNA analysis, and gene expression. Consequently, precise and accurate measurements in various conditions have become more critical for proper result interpretations. Previously, in Part 1, we discussed inner filter effect type I, which is a consequence of the instrumental geometrical sensitivity factor and absorption of the excitation. In this part, we analyze inner filter effect type II and discuss the practical consequences for fluorescence measurements in samples of high optical density (absorbance/scattering). We consider both the standard square and front-face experimental configurations, discuss experimental approaches to limit/mitigate the effect and discuss methods for correcting and interpreting experimental results.

On the origin and correction for inner filter effects in fluorescence Part I: primary inner filter effect-the proper approach for sample absorbance correction.

Fluorescence technologies have been the preferred method for detection, analytical sensing, medical diagnostics, biotechnology, imaging, and gene expression for many years. Fluorescence becomes essential for studying molecular processes with high specificity and sensitivity through a variety of biological processes. A significant problem for practical fluorescence applications is the apparent non-linearity of the fluorescence intensity resulting from inner-filter effects, sample scattering, and absorption of intrinsic components of biological samples. Sample absorption can lead to the primary inner filter effect (Type I inner filter effect) and is the first factor that should be considered. This is a relatively simple factor to be controlled in any fluorescence experiment. However, many previous approaches have given only approximate experimental methods for correcting the deviation from expected results. In this part we are discussing the origin of the primary inner filter effect and presenting a universal approach for correcting the fluorescence intensity signal in the full absorption range. Importantly, we present direct experimental results of how the correction works. One considers problems emerging from varying absorption across its absorption spectrum for all fluorophores. We use Rhodamine 800 and demonstrate how to properly correct the excitation spectra in a broad wavelength range. Second is the effect of an inert absorber that attenuates the intensity of the excitation beam as it travels through the cuvette, which leads to a significant deviation of observed results. As an example, we are presenting fluorescence quenching of a tryptophan analog, NATA, by acrylamide and we show how properly corrected results compare to the initial erroneous results. The procedure is generic and applies to many other applications like quantum yield determination, tissue/blood absorption, or acceptor absorption in FRET experiments.

On the possibility of direct triplet state excitation of indole.

We studied the luminescence properties of indole in poly (vinyl alcohol) (PVA) film. The indole molecules are effectively immobilized in this polymer film and display both fluorescence and phosphorescence emission at room temperature. We noticed that the phosphorescence of indole in PVA film can be effectively excited at a longer wavelength than its typical singlet to triplet population route involving intersystem crossing. The maximum of the phosphorescence excitation is about 410 nm which corresponds to the energy of indole's triplet state. Interestingly, the phosphorescence anisotropy excited with the longer wavelength (405 nm) is positive and reaches a value of about 0.25 in contrast to the phosphorescence anisotropy excited within the indole singlet absorption spectrum (290 nm), which is negative. Very different temperature dependences have been observed for fluorescence and phosphorescence of indole in PVA film. While fluorescence depends minimally, the phosphorescence decreases with temperature dramatically. The fluorescence lifetime was measured to be a single component 4.78 ns while the intensity weighted average phosphorescence lifetime with 290 nm and 405 nm excitations were 6.57 and 5.62 ms, respectively. We believe that the possibility of the excitation of indole phosphorescence in the blue region of visible light and its high anisotropy opens a new avenue for future protein studies.

Non-fluorescent filters for fluorescence detection with in-line geometry.

Intrinsic emission from typical filters can unexpectedly contribute to the total measured signal in a fluorescence system. This emission becomes even more problematic for in-line geometry measurements where the excitation light can directly excite the emission filter. Potassium dichromate has minimal intrinsic fluorescence even with ultra-violet (UV) excitation. We show that a liquid sample can be tuned for its transmission properties by adding a base to the solution. This makes it attractive for use as an optical filter. In addition, when embedded in poly (vinyl alcohol) (PVA) film, potassium dichromate is even less fluorescent. These films can be cut and molded into practically any size or shape. Thus non-fluorescent, modular and cheap filters composed of potassium dichromate embedded in a PVA film is proposed to eliminate this unwanted emission and is suitable for a wide variety of devices and applications.

Identification of Binding Sites for Efflux Pump Inhibitors of the AcrAB-TolC Component AcrA.

The overexpression of multidrug efflux pumps is an important mechanism of clinical resistance in Gram-negative bacteria. Recently, four small molecules were discovered that inhibit efflux in Escherichia coli and interact with the AcrAB-TolC efflux pump component AcrA. However, the binding site(s) for these molecules was not determined. Here, we combine ensemble docking and molecular dynamics simulations with tryptophan fluorescence spectroscopy, site-directed mutagenesis, and antibiotic susceptibility assays to probe binding sites and effects of binding of these molecules. We conclude that clorobiocin and SLU-258 likely bind at a site located between the lipoyl and ß-barrel domains of AcrA.

Enhanced DNA detection using a multiple pulse pumping scheme with time-gating (MPPTG).

Fluorescence signal enhancement induced by the binding of intercalators to DNA has been broadly utilized in various DNA detection methods. In most instances the increase in fluorescence intensity is associated with a concomitant increase of fluorescence lifetime. This increase of the fluorescence lifetime presents an additional opportunity to increase detection sensitivity. In this paper, we present a new approach to significantly enhance the sensitivity in detecting minute DNA concentrations. The approach is based on simultaneous use of time-gated detection and multi-pulse pumping. By using a calibrated burst of short pulses we greatly enhance the contribution of long-lived fluorescence species, thus enabling easy time-gated detection. Using a classic DNA intercalator - Ethidium Bromide (EtBr) - as an example with our novel multi-pulse pumping and time-gated detection technique, we were able to increase detection sensitivity over 70-fold with only 3 pulse excitation. This approach is generic and can be used with any analytical probe (exhibiting about 10 times change in lifetime) that shows an increase in fluorescence signal and fluorescence lifetime upon binding to a target.

Photophysical characterization of anticancer drug valrubicin in rHDL nanoparticles and its use as an imaging agent.

Nanoparticles are target-specific drug delivery agents that are increasingly used in cancer therapy to enhance bioavailability and to reduce off target toxicity of anti-cancer agents. Valrubicin is an anti-cancer drug, currently approved only for vesicular bladder cancer treatment because of its poor water solubility. On the other hand, valrubicin carrying reconstituted high density lipoprotein (rHDL) nanoparticles appear ideally suited for extended applications, including systemic cancer chemotherapy. We determined selected fluorescence properties of the free (unencapsulated) drug vs. valrubicin incorporated into rHDL nanoparticles. We have found that upon encapsulation into rHDL nanoparticles the quantum yield of valrubicin fluorescence increased six fold while its fluorescence lifetime increased about 2 fold. Accordingly, these and potassium iodide (KI) quenching data suggest that upon incorporation, valrubicin is localized deep in the interior of the nanoparticle, inside the lipid matrix. Fluorescence anisotropy of the rHDL valrubicin nanoparticles was also found to be high along with extended rotational correlation time. The fluorescence of valrubicin could also be utilized to assess its distribution upon delivery to prostate cancer (PC3) cells. Overall the fluorescence properties of the rHDL: valrubicin complex reveal valuable novel characteristics of this drug delivery vehicle that may be particularly applicable when used in systemic (intravenous) therapy.

A triazine-based BODIPY trimer as a molecular viscometer.

Photophysical behaviour of a novel trimeric BODIPY rotor with a high extinction coefficient is reported. Steady state and time resolved fluorescence measurements established that the trimer could be used as a viscometer for molecular solvents, membrane-like environments and several cancer cell lines.

BODIPY-BODIPY dyad: assessing the potential as a viscometer for molecular and ionic liquids.

A symmetrical BODIPY-BODIPY dyad with a diyne linker was prepared in two steps; the lifetime decay of this rotor appeared to correlate with the viscosity of the media, thus making this dyad a suitable small molecule viscometer for molecular solvents. The potential of using the rotor to probe the viscosity of ionic liquids was also investigated.

Physicochemical properties of potential porphyrin photosensitizers for photodynamic therapy.

This research evaluated the suitability of synthetic photosensitizers for their use as potential photosensitizers in photodynamic therapy using steady state and time-resolved spectroscopic techniques. Four tetraphenylporphyrin derivatives were studied in ethanol and dimethyl sulfoxide. The spectroscopic properties namely electronic absorption and emission spectra, ability to generate singlet oxygen, lifetimes of the triplet state, as well as their fluorescence quantum yield were determined. Also time-correlated single photon counting method was used to precisely determine fluorescence lifetimes for all four compounds. Tested compounds exhibit high generation of singlet oxygen, low generation of fluorescence and they are chemical stable during irradiation. The studies show that the tested porphyrins satisfy the conditions of a potential drug in terms of physicochemical properties.

A homodimeric BODIPY rotor as a fluorescent viscosity sensor for membrane-mimicking and cellular environments.

Fluorescence properties of a novel homodimeric BODIPY dye rotor for Fluorescence Lifetime Imaging Microscopy (FLIM) are reported. Steady state and time resolved fluorescence measurements established the viscosity dependent behaviour in vitro. Homodimeric BODIPY embedded in different membrane mimicking lipid vesicles (DPPC, POPC and POPC plus cholesterol) is demonstrated to be a viable sensor for fluorescence lifetime based viscosity measurements. Moreover, SKOV3 cells readily endocytosed the dye, which accumulated in membranous structures inside the cytoplasm thereby allowing viscosity mapping of internal cell components.